Transcriptome Analysis Of The Halostachys Caspica Under Salt Stress And Identification Of The Functional Gene

Soil salinization,as one of the main abiotic stress factors affecting crop production,not only exacerbates the deterioration of the ecological environment,but also brings hidden dangers for food production security.High concentration of salt will produce secondary damage to the plant including osmotic stress,oxidative stress and ion toxicity,which will affect the yield and quality of crops.As the largest saline-alkali soil distribution area in China,Xinjiang has an area of 110,000 square kilometers of soil salinization,which accounts for 7% of the total area of Xinjiang.However,in the saline-alkaline environment of Xinjiang,there are numerous types of halophytes.Therefore,indepth study of the molecular mechanisms under salt tolerance of halophytes,screening and excavation of salt-tolerant functional genes will help improve the salt tolerance of plants using genetic engineering techniques,and cultivate new varieties of crops with high salt tolerance,which will become important technical means for the development and utilization of saline-alkali land.However,due to the poor understanding of the salt-tolerant molecular mechanism of plants and the lack of effective salt-tolerant genes,the application and development of plant salt-tolerance genetic engineering technology has been slowly developing.Halostachys caspica(Chenopodiaceae,Halostachys)is a shrub with extreme salt tolerance.It widely distributes in the desert saline area,and has important ecological value.In the process of long-term evolution,it has adapted to salt stress with unique characteristics and regulatory mechanisms.High-throughput sequencing technology was used to detect the transcriptome of Halostachys caspica under salt stress.It is a good way to study the differential gene expression of Halostachys caspica in response to salt stress.In this paper we selected Halostachys caspica as study material to carry out our salt stress research.(1)The transcriptome of Halostachys caspica treated with 500 mmol/L NaCl for0 hour(control),3 hours(short-term treatment)and 15 days(long-term treatment)was conducted using RNA sequencing(RNA-seq)on the Illumina Hi Seq TM 4000 platform.A transcriptome database of 88,574 unigenes with an N50 length of 997 bp were assembled using Trinity.4,606 and 7,088 genes were significantly differentially expressed between short-term and long-term treatment,respectively.Gene ontology(GO)analysis revealed 47 highly enriched terms,and a differential response of metabolic pathways between the short-term and long-term treatment.Kyoto Encyclopedia of Genes and Genomes(KEGG)annotation revealed that the DEGs were highly enriched in Plant hormone signal transduction(ko04075),Phenylpropanoid biosynthesis(ko00940),MAPK signaling pathway-plant(ko04016)in both salt treatments.Finally,the transcriptome results were verified by qRT-PCR technique and physiological biochemical indicators.The results showed that the transcriptome sequencing results were accurate and reliable.(2)Under short-term salt stress,compared with the control,it was found that betaine,soluble sugar and POD activity were significantly increased,while Pro was decreased,but under long-term salt stress,POD and osmoregulation related to betaine and soluble sugars were significantly increased,while Pro was slightly increased.Under long-term salt stress,the ROS in the Halostachys caspica accumulated and the osmotic stress increased.Therefore,the plant needs to synthesize a large number of ROS scavenging enzymes and osmotic adjustment-related substances to resist salt stress.(3)According to the HcUKPP gene obtained from the previous clone,the expression of HcUKPP was detected at 3 h and 15 d after salt stress,indicating that HcUKPP was induced by salt stress.The recombinant strain Escherichia coli BL21::pET30a-HcUKPP was obtained by the method of prokaryotic vector construction after cloning of HcUKPP,and then tested its tolerance under different abiotic stresses.The growth of recombinant strain E.coli BL21::pET30a-HcUKPP have shown obvious advantages under treatment of different concentrations of NaCl(100～900 mmol/L),polyethylene glycol(2.5%～20%,PEG 6000)and methyl viologen(25～200 μmol/L).Especially under the condition of 500 mmol/L NaCl、10% PEG6000 and 75 μmol/L methyl viologen for 12 h,interestingly,recombinant E.coli BL21 showed significant advantages,their growth reached 1.81-fold,1.47-fold and3.48-fold of control bacteria respectively.Construction of plant expression vector pCAMBIA1301-HcUKPP,and genetic transformation Arabidopsis thaliana.Overall,the HcUKPP gene of Halostachys caspica can significantly improve tolerance of recombinant E.coli BL21 under different abiotic stress,which proved that the HcUKPP is a kind of newly discovered polypeptide in response to abiotic stress.